Differentiation of promyelocytic HL-60 cell line into primed neutrophils for the evaluation of antiapoptotic effects of poorly soluble nanoparticles
Description
Neutrophil apoptosis is an important determinant of intensity and duration of neutrophilic inflammation. The interaction of poorly soluble nanoparticles with primed neutrophils can reduce their natural apoptosis rates. This reaction may contribute to pathogenic consequences of increased neutrophilic inflammation. Toxicological studies aiming to identify hazards of such materials with primary neutrophils are however challenging due to the short life span of these cells and a high donor to donor variability. Our purpose was the establishment of a culturable neutrophil-like cell line as a suitable model for studies of antiapoptotic effects of poorly soluble combustion-derived environmental nanoparticles. Therefore, differentiation protocols for the promyelocytic leukemia HL-60 cell line based on commonly used differentiation inducers all-trans retinoic acid (ATRA) and dimethyl sulfoxide (DMSO) were established and compared. The data demonstrate that only a combined cell treatment with ATRA and DMSO for a period of 5 days leads to the complete HL-60 differentiation with the typical neutrophil morphology and characteristic features of neutrophil maturation like cell cycle arrest, increase in differentiation marker CD11b, loss of proliferation marker CD71, and increased phagocytic capacity. Exposure of cells differentiated with ATRA + DMSO to carbon nanoparticles or proinflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) revealed a delay of apoptosis causally linked to intracellular reactive oxygen species (ROS). These data verified our earlier findings with human peripheral primed neutrophils from donors with slightly elevated proinflammatory blood plasma factors. Moreover, completely differentiated HL-60 cells possessed similar levels of L-selectin CD62L as neutrophils with primed immunophenotype, thus representing the biology of primed inflammatory neutrophils. Neutrophil-like HL-60 cells differentiated according to our protocol could be an appropriate substitute cell line model for studies on the effects of inhalable nanomaterials on primed inflammatory neutrophils like lung neutrophils. For such toxicological studies our cell model is preferable to peripheral neutrophils as blood neutrophils not always occur in a primed state and primed lung neutrophils from donors are not available for this purpose.
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Steps to reproduce
In order to develop a robust and reproducible neutrophilic cell model to determine the antiapoptotic effect of inhalable materials, we investigated the impact of ATRA and DMSO each alone or in combination at different time points of treatment. The success of the differentiation was tested by monitoring of cell morphology (with May-Guenwald/Giemsa staining) and characteristic changes of neutrophil maturation like proliferation and cell cycle, apoptosis, phagocytic capacity, as well as cell surface markers of differentiation and priming. To analyze HL-60 proliferation, cells were counted microscopically at day 0 and on every day of the differentiation period using the trypan blue dye exclusion test. Phagocytic capacity of HL-60 cells was analyzed as uptake of pH-sensitive fluorescence-labelled bioparticles (Thermo Fischer Scientific #35367) by flow cytometry. For cell cycle analysis and apoptosis measurement (% sub-G1), HL-60 cells were stained according to Nicoletti protocol by direct DNA staining in propidium iodide hypotonic solution and flow cytometry. Apoptosis in HL-60 cells was verified with Annexin V-FITC/PI staining according to the instructions of the manufacturer (BioLegend #640914). Cell surface markers of differentiation (CD11b, CD71) were analyzed using specific antibodies (BioLegend) and flow cytometry.
Institutions
- Leibniz-Institut fur umweltmedizinische Forschung an der Heinrich-Heine-Universitat Dusseldorf gGmbH